Enzymes and microorganisms, through the direct reduction of molecular oxygen, naturally catalyze wide ranges of oxidation reactions. These highly irreversible reactions can be regarded as quite violent from a biochemical perspective liberating much free energy of reaction as heat. The fundamental mechanism by which the many diverse biooxidations of organic substrates occur can generally be regarded as, first, the activation of molecular oxygen to form a highly reactive oxygen species. This is followed by a shift of electrons from the substrate to the activated oxygen species resulting in the net oxidation of the substrate. The original oxygen atoms from molecular oxygen may or may not be incorporated into the organic substrate to make the desired oxidation product. The reaction may be catalyzed by a single enzyme or through the coordinated action of multiple enzymes. Many enzyme and enzyme systems comprising, cytochromes, monooxygenases, dioxygenases, desaturases, and oxidases are capable of harnessing the reactive power of molecular oxygen for the oxidation of organic substrates with a selectivity and specificity unparalleled by classical chemical synthesis.
There is much incentive to exploit these reactions by applying them to ordinary chemical substrates by adding one or more chemical functionalities at particularly desirable positions in the substrate molecule. Owing to the selectivity and specificity of certain biocatalysts, many chemical reactions and purification steps with their concomitant product and substrate losses can be eliminated. Alternatively, these reactions can aid the degradation of unwanted chemical products, again by adding functionality to the molecule making it easier to sequester for disposal or degrade further in subsequent transformation steps.
There are many barriers to overcome in successfully operating these reactions for commercial purposes. The generation of highly reactive oxygen species is, in metabolic terms, a violent reaction. These reactive intermediates can damage or degrade the biocatalyst, destroy the enzymatic activity sought for making the desired transformation, or react non-selectively with the substrate. As pointed out by Stryer in his text “Biochemistry”—Danger lurks in the reduction of oxygen. Organisms utilizing molecular oxygen possesses enzymes and enzyme systems for scavenging renegade oxygen radicals in nature. In the rapid biooxidation reactions occurring in industrial fermentations, such radicals may overwhelm the naturally occurring levels of these scavenging systems. As a result, in biooxidation reactions involving viable cells, it is common to observe a rapid decay in cell viability and the recovery of viable cells (Dixon, B., “Viable but Nonculturable”, ASM News, 64, pgs. 372-373, 1998).
Wide ranges of organic molecules are potential substrates for bio-oxidation, without limitation to the natural substrate preference of the oxidation pathway, to make many oxidation products. Certain characteristics of the substrate may dictate how it will be combined with the oxidation catalyst to effect the oxidation. The substrate may    1) be toxic, deactivating the catalyst,    2) be volatile and susceptible to losses in the fermentor off-gases,    3) repress its own oxidation,    4) induce or cause unwanted side reactions,    5) be expensive; committing large quantities of substrate to the vessel adds risk should the batch fail through equipment failure or contamination,    6) generate non-selective oxidation reactions or other reactions,    7) cause problems in recovering or purifying the product,    8) may form flammable mixtures with air.
For these reasons, it is often desirable to add the substrate continuously or in discretely metered increments during the fermentation in a fed-batch-operating mode to minimize substrate accumulation.
Despite the large amount of free energy thermodynamically available from many biooxidation reactions, these reactions tend to be highly irreversible liberating much of the free energy of reaction as heat rather than as forms useful for metabolic work. This is due to the initial metabolic energy input required to create the reduced oxygen species from molecular oxygen. To generate this needed metabolic energy driving the desired biooxidation reaction and maintaining the biocatalyst, a second substrate is supplied to the reaction system. This second substrate is frequently called the co-substrate and may be selected from any of a number of fermentable compounds in common use for fermentation, for example, sacharrides, organic acids, alcohols, or hydrocarbons. As for similar reasons with the bio-oxidation substrate, it may be desirable to add the co-substrate continuously or in discretely metered increments to minimize accumulation of the co-substrate.
Some control of substrate concentration is known in the art. For example, the concentration of the substrate ML236B Na was controlled in a cytochrome P450 mediated hydroxylation reaction to make pravastatin. A crossflow filtration module and peristaltic pump generated a filtrate. This filtrate was periodically analyzed by automatic HPLC to give near real time measurement of ML236B Na concentration in the aqueous reaction mixture.
The crossflow system worked well with the soluble substrate apparently because the strain did not form large pellets and no oily substances were present in the reaction mixture. No control however was applied to the cosubstrate 50% glucose feed which was maintained constant over the course of the reaction.
Thus there is a need for methods for simultaneously controlling the addition of multiple feed streams in the field of biooxidations. Biooxidation substrates, cosubstrates, and products are frequently complex molecules that may be sparingly soluble in aqueous systems that, together with the biocatalyst, constitute a complex reaction matrix. While a variety of analytical techniques may be applied offline for the analysis of products, substrates, and co-substrates, such methods tend to be laborious and of little value for real-time control of nutrient feed in these fed-batch reactions. One is therefor motivated to seek rapid computerized methods for simultaneously controlling the substrate and cosubstrate feeds to biooxidation reactions.
In contrast, the gas phase is a relatively simple matrix for which a variety of instruments are available to give gas phase composition measurements. When coupled with a measurement of gas flow to and from the biooxidation reaction, the overall and component gas phase balances provide real time measurement of carbon dioxide evolution rate (CER) and oxygen uptake rate (OUR).
The concept of controlling nutrient feeds in fed batch fermentations using component gas balances is known in the art. In particular, gas component balance measurements have been used to calculate and control substrate concentration or to control specific growth rate.